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Rochalimaea quintana, the etiological agent of trench fever, was tested by an agar dilution method for its susceptibility to the following 14 antibiotics: penicillin G, methicillin, ampicillin, cephalothin, vancomycin, doxycycline, tetracycline, erythromycin, chloramphenicol, streptomycin, kanamycin, rifampin, colistin, and amphotericin B. The MIC of each of these antibiotics was determined. The results showed that R. quintana is susceptible in vitro to these antibiotics, with the exception of vancomycin, kanamycin, streptomycin, colistin, and amphotericin B.

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The systematic study ofLegionella as a human pathogen and a bacterium widely disseminated in the environment requires simplification of present methodology. We describe a highly sensitive enzyme-linked immunosorbent assay (ELISA) for the detection of serum antibodies that can also be used for the detection of antigen.Legionella pneumophila serogroups 1 and 3 (Philadelphia 2 and Bloomington 2),L. bozemanii (WIGA), andL. micdadei (TATLOCK) were grown in diphasic medium consisting of charcoal yeast extract agar (CYE) overlayed with yeast extract medium (YEM) for the production of whole cell antigen and CYE for the extraction of carbohydrate antigen. The whole cells were inactivated with 0.5% formalin. The carbohydrate was obtained from the supernatant of cells resuspended twice in phosphate buffered saline (PBS). The antigen was sterilized and concentrated by filtration and purified by chromatography through a Sepharose 4B column. The highest molecular weight fractions were used for chemical characterization, which confirmed the carbohydrate nature of the antigen, and for micro-ELISA. Titers ranging from 5×10(3) to 3×10(5) (inverse of serum dilutions) were obtained from rabbit sera collected after 1, 2, or 3 injections of whole cells. The titers were somewhat higher and more consistent with the higher of 2 antigen concentrations used (5 or 15µg/ml protein or dry weight), and with the carbohydrate rather than the whole cell antigen. The reactions were serogroup and species specific and only low titers were obtained with some of the heterologous antigens. The sensitivity and specificity of the reactions were not diminished when as many as 4 antigens were mixed in the same well. Thus, the micro-ELISA can be used as a test of highly specific antigens as well as a screening test with mixtures of antigens. A preliminary test withLegionella containing water specimen concentrates and high-titer rabbit sera indicated that the micro-ELISA can also be used for the detection of antigen. This investigation appears to have paved the way for the simplification of the serological methodology for the study ofLegionella.

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The genome size of Coxiella burnetii Nine Mile strain was determined by the method of initial rate of deoxyribonucleic acid renaturation. The mean value obtained was 1.04 X 10(9) daltons.

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We investigated the recent claim that the vole agent, a rickettsia-like microorganism isolated from wild voles by Baker in 1946, is actually a strain of Rochalimaea quintana, the etiological agent of trench fever. The two organisms were compared on the basis of percent guanine-plus-cytosine content, genome size, deoxyribonucleic acid-deoxyribonucleic acid hybridization, polypeptide composition, and serological relationships. Although the two organisms do have identical or nearly identical deoxyribonucleic acid base ratios and show some serological cross-reactions, they differ substantially by all of the other criteria employed. They are clearly different, although possibly related, organisms. It remains to be determined whether they can be regarded as two species of the same genus. On the other hand, an Old World strain and a New World strain of R. quintana were indistinguishable from one another by the same criteria.

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Glutamic acid oxidation by Rickettsia prowazeki is not accompanied by hydrogen peroxide generation, nor is added hydrogen peroxide degraded, as measured by a manometric technique.

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No bactericidal effect was produced when Rochalimaea quintana was exposed for 1 h to a combination of high-titered anti-R. quintana rabbit serum and guinea pig complement.

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Rickettsia quintana grew in a liquid medium consisting of a brain-heart infusion base supplemented with starch and hematin. The growth requirement for hematin could not be substituted by compounds of known catalytic activity for H(2)O(2), viz., catalase, potassium pyruvate, or charcoal, or by the reducing compounds sodium sulfite and sodium thioglycollate. R. quintana was catalase-negative, but no H(2)O(2) production could be demonstrated by the catalase-aminotriazole technique. A minimum inoculum giving 10(5) cells/ml was required to initiate growth. The generation time at 33 C was 10 hr. The temperature range for growth was 28 to 37 C. Growth was enhanced when succinate or glutamate was added as energy source.

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Rickettsia quintana grew readily on blood-agar base when the following conditions and supplements were supplied: (i) aerobic conditions; (ii) increased CO(2) tension; (iii) crystalline hemoglobin or hemin, but not protoporphyrin; and (iv) a colloidal "detoxifying agent," such as starch or charcoal. Serum was not required nor did it enhance growth when all of the aforementioned components were supplied.

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The passive permeability properties of Rickettsia mooseri to both inorganic and organic solutes have been examined. Visual observations by phase-contrast microscopy of rickettsiae in macerated yolk sacs taken directly from heavily infected eggs revealed plasmolysis with hypertonic NaCl and KCl as well as with sucrose solutions. In contrast, similar visual studies of rickettsiae which had been subjected to freezing or to a purification process, or both, were plasmolyzed by hypertonic sucrose but not by hypertonic NaCl and KCl. These primary observations were extended to a variety of solutes and were placed on a quantitative basis by use of optical density and radioisotope dilution methods. Intracellular Na(+) and K(+) concentrations in processed rickettsiae, measured by flame photometry, closely paralleled the concentration of these ions in the suspending medium. It was concluded that R. mooseri appears to possess an osmotically active, functional, and structural membrane distinct from the cell wall, located at the surface of a structure analogous to the bacterial protoplast. In the intact organism, this membrane is passively impermeable to sucrose, NaCl, and KCl. However, altered permeability properties, especially to inorganic electrolytes, may be expected in rickettsiae which have been stored in the frozen state and subjected to a lengthy purification process.

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